The Journal of Experimental Biology 200, 3033–3041 (1997) 3033 Printed in Great Britain © The Company of Biologists Limited 1997 JEB1117

DIFFERENT EXCITATION–CONTRACTION COUPLING MECHANISMS EXIST IN , AND OCTOPOD MUSCLE

C. M. ROGERS1, L. NELSON2, B. J. MILLIGAN1 AND E. R. BROWN1,* 1Marine Biological Association of the UK, Citadel Hill, Plymouth PL1 2PB, UK and 2Christ’s College, University of Cambridge, Cambridge CB2 3BU, UK

Accepted 1 September 1997

Summary Excitation–contraction (EC) coupling was studied in voltage-clamp experiments on isolated muscle fibres from central zone mantle muscle fibres of a squid (Alloteuthis squid, cuttlefish and Eledone cirrhosa, a sustained inward subulata), a cuttlefish (Sepia officinalis) and an octopod current was recorded upon depolarisation. This current (Eledone cirrhosa). Thin slices of muscle were used for was blocked by 5 mmol l−1 Co2+ and suppressed by twitch experiments and enzymatic isolation of single fibres 10 µmol l−1 nifedipine. In squid, an additional inward fast- for whole-cell patch-clamp studies. The current required activating transient current was seen which was blocked by for a supramaximal twitch response during direct 2 µmol l−1 tetrodotoxin and depolarised holding potentials. stimulation of muscle slices was lower for squid than for The fast current represents a voltage-activated Na+ cuttlefish. In squid, but not in cuttlefish, the channel, and the slow currents represent L-type Ca2+ current–response relationship was independent of slice channels. We conclude that squid possess a specialised thickness (range 0.1–0.5 mm). Twitches of squid and rapid EC coupling mechanism in central zone fibres that is cuttlefish slices were reversibly abolished by removal of absent in cuttlefish and Eledone cirrhosa. extracellular Ca2+. In squid, but not in cuttlefish, the current–response relationship was Na+-dependent, and in Key words: squid, Alloteuthis subulata, Ca2+ channel, Na+ channel, the absence of Na+ higher current strengths were required excitation–contraction coupling, cuttlefish, Sepia officinalis, octopus, to generate a supramaximal response. In whole-cell Eledone cirrhosa, mantle muscle.

Introduction The mantle performs many functions including to be fully established, but they are likely to be aerobic since slow swimming, fast swimming, escape jetting and respiration they are involved in the inspiratory phase of the respiratory (Bone et al. 1994). The mantle contains at least two cycle (Bone et al. 1994). It is not known whether these orientations (radial and circular; see Fig. 1) of antagonistic, morphological and biochemical differences are reflected in the obliquely striated muscle and, in cuttlefish and squid, is membrane physiology of these muscle fibre types. innervated by giant and small-diameter . However, the Many attempts have been made to determine the functional contributions of the precise pattern of innervation and the innervation of the mantle muscle (Prosser and Young, 1937; physiology of the muscle fibres to mantle function are unclear Young, 1938; Wilson, 1960; Brown et al. 1991), and it seems (Bone et al. 1995). likely that the inner and outer fibres receive their innervation Contraction of the radial fibres thins the mantle wall, thereby from small-diameter axons. It is not clear at present, however, increasing mantle diameter and drawing water into the mantle whether central zone fibres receive dual or parallel innervation cavity. Conversely, contraction of the circular fibres reduces from the giant and small-diameter axons. Current evidence mantle diameter and expels water from the mantle cavity via favours parallel innervation, as escape jets may be driven by the exhalant . The circular fibres may be subdivided into giant and/or by small-diameter axons (Otis and Gilly, 1990). two types (Fig. 1) on the basis of their biochemistry and While the organisation of muscle fibres within the mantle has morphology. Mitochondria-rich fibres form thin inner and outer been established, few investigations have directly addressed layers, which delimit an extensive central zone of mitochondria- muscle fibre physiology. The mechanical properties of fibres poor fibres (Bone et al. 1981; Mommsen et al. 1981). It has from squid and cuttlefish were described in recent studies that been suggested that the central zone operates anaerobically and used a slice preparation to isolate central and peripheral fibre the outer and inner fibres operate aerobically (Bone et al. 1995). types (see preliminary experiments by Usher, reported in Bone The biochemistry and morphology of the radial fibres has yet et al. 1994; Milligan et al. 1997). The difficulty in obtaining

*Author for correspondence (e-mail: [email protected]). 3034 C. M. ROGERS AND OTHERS

I C Cz C O Fig. 1. Diagram of the arrangement of muscle fibres (not to scale) in the squid C S (and cuttlefish) mantle, showing the circular (C) fibres consisting of inner (I), R outer (O) and central zone (Cz) fibres and R the radial (R) fibres. The plane of section (S) made by the vibratome slice is also R R shown. suitable preparations of these small fibres (8–10 µm in (Sepia officinalis L.) and the octopod (Eledone cirrhosa L.) diameter) has greatly hindered serious electrophysiological were trawled off Plymouth, UK, and kept at the Marine studies. Brown (1991), cited in Bone et al. (1995), described Biological Association Laboratory in circulating sea water at rapid overshooting spike potentials, from resting potentials of 10–15 °C. The animals were killed by decapitation under deep approximately −70 mV, in central mantle fibres of the small ethanol anaesthesia, and a piece of the ventral mantle wall was squid Alloteuthis subulata. More recently, viable single fibres dissected from the long axis of the mantle, in the region of the were dissociated from the mantle of juvenile squid Loligo mid-line. opalescens and examined using the voltage-clamp technique Small pieces (1 cm×1 cm) of mantle from Alloteuthis and (Gilly et al. 1996). A voltage-activated Na+ current was Sepia were sliced using a vibratome into 0.1 mm horizontal identified in a population of cells representing approximately sections (see Fig. 1). During slicing, the tissue was immersed 50 % of the fibres sampled. As it was not possible in that study in, EGTA-buffered Ca2+-free artificial sea water (Ca2+-free −1 to establish from which mantle zones the muscle fibres had ASW) containing (in mmol l ): NaCl, 450; MgCl2, 10; EGTA, come, it remains to be seen whether the giant (fast) and small- 10; Hepes, 10; pH 7.8 (4–5 °C). Full details of this procedure diameter (slow) axons are complemented by fast (Na+- have been described previously (Milligan et al. 1997). Care activated) and slow (Ca2+-activated) muscle fibres. In addition, was taken to isolate slices specifically from the central zone of the question of whether a similar distribution of channels exists mitochondria-poor fibres. The mantle muscle of Eledone was in muscle fibres from the ‘slower’ benthic relatives of the squid, gelatinous in consistency and was impossible to slice with a the cuttlefish and octopods, remains to be answered. vibratome. A piece of the muscle was therefore sliced We have used the slice technique to obtain central zone manually into sections approximately 0.1 mm thick. The muscle fibres to answer some of these questions, using muscle slices were stored in EGTA-buffered Ca2+-free ASW biomechanical measurements and the whole-cell configuration (4–5 °C) until required. of the patch-clamp technique. Our results confirm that Na+ channels are present in squid mantle muscle and establish that Mechanical experiments they are located on central zone fibres. Na+ channels are absent Rectangular preparations, approximately 10 mm in length on equivalent zones from cuttlefish and octopod mantle, and and 5 mm in width, were dissected from the vibratome slices all three species possess L-type Ca2+ channels. The results under cooled (4–5 °C) Ca2+-free ASW. T-shaped aluminium have implications for our understanding of mantle physiology foil clips were used to attach one end of the preparation to a and of the phylogeny of EC coupling. force transducer (type ST01, Devices) and the other end to a stiff glass rod. Force produced during muscle contraction was recorded isometrically. The tissue was continuously superfused Materials and methods with a thin film of cooled (12±0.5 °C), aerated artificial sea Animals water containing glucose (g-ASW) consisting of (in mmol 1−1): Adult squid (Alloteuthis subulata Lamarck), cuttlefish NaCl, 470; KCl, 10; CaCl2, 10; MgCl2, 50; glucose, 20; Hepes, EC coupling in cephalopod mantle muscle 3035

10; pH 7.8. The preparation was stimulated using rectangular artificial sea water (nCa2+-free ASW) containing (in mmol l−1): current pulses (model DS7 stimulator, Digitimer Ltd, UK) 470 NaCl, 9 KCl, 10 MgCl2 and 10 Hepes, pH 7.8, and delivered via large platinum plate electrodes aligned parallel to dissociated in 4 mg ml−1 Papain and Collagenase P (Sigma) in the long axis of the tissue slice. Pulse duration was 2 ms, and nCa2+-free ASW for 1 h at room temperature (21 °C). The stimulation interval was 60 s. The stimulation pattern and force tissue was washed in nCa2+-free ASW and then agitated in a were controlled and recorded using a ViewDac (Keithley, UK) culture dish containing four coverslips in nCa2+-free ASW. sequence via a data acquisition board (Lab Master DMA, The coverslips were then stored at 15 °C for up to 5 h. Only Scientific Solutions Inc., USA). myocytes that adhered to the coverslips were used for voltage- Initially, muscle length was adjusted such that a transient clamping, using the whole-cell voltage-clamp method. resting tension was just apparent. The stimulus intensity–twitch response relationship was then investigated Solutions during superfusion of g-ASW. Frequent control stimulations of In order to reduce rundown of currents by internal perfusion constant current amplitude were given to monitor the degree and contracture of fibres in solutions of high ionic strength, and of decline of twitch force as the trial progressed. The to maintain iso-osmolarity, internal and external solutions were current–response relationship was then established for a diluted to one-third concentration using a 12 % glycerol solution second time, during superfusion of g-ASW with the addition (see Inoue et al. 1994). The external solution, used for measuring of a glutamate antagonist, 2-amino-4-phosphonobutyric acid inward Ca2+ and Na+ currents was one-third g-ASW (without (2-APB), at a concentration of 20 µmol l−1. This concentration glucose) and two-thirds 12 % glycerol, pH 7.8. The internal − was sufficient to inhibit glutamate contractures during solution contained one-third 450 mmol l 1 caesium D-aspartate, −1 −1 −1 preliminary trials with each preparation (see also Milligan et 15 mmol l MgCl2, 30 mmol l EGTA–Cs and 10 mmol l al. 1997). In all but two squid preparations, data from which Mops–Cs, pH 7.2, and two-thirds 12 % glycerol. Nifedipine was were subsequently discarded, the addition of 2-APB did not dissolved in absolute ethanol to make a 0.5 mmol l−1 stock significantly modify the current–response relationship, solution which was stored at 2 °C in the dark. 2 µmol l−1 indicating that the muscle cells were stimulated directly. tetrodotoxin (TTX) and 1 mol l−1 Co2+ stock solutions were Stimulus current was adjusted to a level 50 % higher than made up in distilled water and added to the bath to block Na+ that required to elicit a maximal twitch response. The and Ca2+ currents, respectively. All chemicals were purchased length–force relationship of the preparation was then from Sigma, and experiments were carried out at 12–13 °C. established for twitch stimulation. At the end of a series, the length of the muscle was adjusted to that giving peak twitch Whole-cell voltage-clamp force (Ltw). The stimulus current–twitch response relationship The coverslips holding the myocytes were transferred to a was determined anew at Ltw. Measurements were then repeated culture dish held in the stage of an inverted microscope during superfusion of either Ca2+-free ASW or Na+- free (Nikon). Electrodes were pulled with a Flaming–Brown puller artificial sea water (Na+-free ASW) containing (in mmol 1−1): to resistances of 1–2 MΩ. Whole-cell patch-clamp experiments N-methyl-D-glucamine, 470; KCl, 10; CaCl2, 10; MgCl2, 50; were conducted using a laboratory-made amplifier with a head- glucose, 20; Hepes, 10; pH 7.8. A final control trial during stage feedback resistor of 1 GΩ. The series resistance of superfusion of g-ASW was also recorded. In some approximately 1 MΩ was compensated. Pulse generation and experiments, the current–response relationship at Ltw was detection were achieved using 12-bit D/A and A/D converters determined for muscle slices over a range of slice thicknesses (Scientific Solutions Inc., Labmaster DMA, USA) and an (0.1, 0.3 and 0.5 mm). IBM/AT computer using P-Clamp software ( At the end of an experiment, the length of the preparation at Instruments, USA). Linear cell membrane capacitance (Cm) Ltw was measured with an eyepiece micrometer under a was measured by integrating the area under a transient dissecting microscope. The preparation was fixed (5 % capacitative current produced by a 10 mV step depolarisation glutaraldehyde in 75 % g-ASW, buffered with 0.5 mol l−1 from a holding potential of −70 mV. sodium cacodylate), dried to constant mass at 70 °C, and weighed using a microbalance. Specific force was expressed as Data acquisition and analysis mN mm−2 cross sectional area (CSA) of tissue. Area was In each recording of current, linear components (except for calculated as volume/length, with volume calculated from the membrane capacity measurements) were cancelled (using the dry mass of the tissue (assuming a density of 1.06 g cm−3; P/4 method; see Inoue et al. 1994) by adding five traces Mendez and Keys, 1960) and the wet mass:dry mass ratio associated with four control pulses and one test pulse. The (determined for cuttlefish mantle muscle as 4.14; Milligan et control pulses had one-quarter amplitude and the opposite al. 1997). polarity to the test pulse and were applied from −70 mV.

Single cell preparation Slices (0.1 mm thick) of mantle muscle were enzymatically Results dissociated in 1 mg ml−1 Trypsin (Type III, Sigma) in Ca2+-free Mechanical experiments ASW for 20 min at 5 °C, washed briefly in nominally Ca2+-free Records of typical isometric twitch responses at Ltw are 3036 C. M. ROGERS AND OTHERS

1.0 Fig. 2. Typical records of supramaximal twitches elicited by direct 0.9 stimulation of muscle slices at Ltw, the muscle length giving peak twitch force. Twitches from both squid and cuttlefish slices are shown, 0.8 with force normalised to the maximum observed force of the 0.7 preparation. Note the rapid activation of the squid muscle. The arrow 0.6 shows the point at which the stimulus was given. 0.5 Cuttlefish Squid 0.4 between stimulus strength and twitch force during direct Relative force 0.3 stimulation of thin-slice preparations (0.1–0.5 mm thickness) is 0.2 summarised in Fig. 3. Data for both squid and cuttlefish 0.1 preparations are shown. Relative force is expressed with 0 respect to the peak isometric twitch force observed during the 100 ms course of a trial. The current required to elicit a supramaximal response was high (range 15 to >100 mA for all experiments), as expected for direct stimulation regimes (also see Milligan shown in Fig. 2. The records show that squid muscle is more et al. 1997). Squid preparations were maximally activated at a rapidly activated than that of cuttlefish. The relationship lower stimulus strength than cuttlefish preparations of similar

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Fig. 3. Relationship between stimulus strength 0.1 mm 0.2 and twitch force showing the effect of slice 0.3 mm thickness on tissue activation. Force is 0.5 mm normalized to the maximum produced by the 0 preparation. Mean values ± S.E.M. for (A) squid muscle [N=9 (0.1 mm), 7 (0.3 mm), 8 (0.5 mm)] − and (B) [N=10 (0.1 mm), 9 (0.3 mm), 0.2 7 (0.5 mm)] cuttlefish muscle. Activation is 0 20406080100120 dependent on slice thickness in cuttlefish alone. Current (mA) EC coupling in cephalopod mantle muscle 3037 thickness. Peak specific isometric force was superfusion of Na+-free ASW (Fig. 4A). Nevertheless, 42.9±3.8 mN mm−2 CSA for preparations from squid (N=18) isometric response properties during supramaximal stimulation and 34.7±5.1 mN mm−2 CSA for preparations from cuttlefish were not significantly modified, and twitch responses were (N=17) (mean ± S.E.M.). In preparations from squid, the quantitatively similar to those observed in g-ASW. In each current–response relationship was found to be independent of preparation studied, normal contractile activity was slice thickness within the range studied (Fig. 3A). In cuttlefish, subsequently restored by superfusion of g-ASW (Fig. 4A). In however, the current required to elicit a supramaximal seven trials on preparations from cuttlefish, superfusion of response in g-ASW was strongly dependent on slice thickness, Na+-free ASW did not significantly affect the current–response increasing as slice thickness increased (Fig. 3B). relationship (Fig. 4B). Because it seemed likely that removal Twitch responses of preparations from both cuttlefish (N=7) of Na+ could block the Na+/Ca2+ exchange mechanism, leading and squid (N=9) were reversibly abolished in Ca2+-free ASW to an increase in resting tension, we checked the records (data not shown). Subsequent superfusion of g-ASW resulted carefully for any changes in resting force during and after in the restoration of normal contractile activity in each superfusion with Na+-free solutions. We detected no increase preparation studied. In Na+-free ASW, however, inter-specific in resting tension during or after superfusion. differences in contractile response were observed. In nine trials on preparations from squid, the current required to elicit a Whole-cell currents supramaximal response was significantly increased during Ionic currents from single myocytes from the mantle of

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Fig. 4. Relationship between stimulus strength 0.2 Normal ASW and twitch force showing the effect of Na+-free Na+-free ASW solutions. Force is normalized to the maximum 0 Normal ASW produced by the preparation. Mean values ± S.E.M. for (A) squid muscle (N=9) and (B) − cuttlefish muscle (N=7). Results are shown in 0.2 standard artificial sea water (g-ASW), Na+-free 0 20406080100120 ASW and after return to g-ASW. Current (mA) 3038 C. M. ROGERS AND OTHERS ) 1 − A D 4 2 − − 60 40 Current (pA pF 20 60 Voltage (mV) −2 Fig. 5. Whole-cell voltage-clamp current records showing Na+ and Ca2+ currents in isolated squid B mantle muscle (A) before and (B) after the application of 2 µmol l−1 tetrodotoxin (TTX), and 600 pA (C) after the application of 5 mmol l−1 Co2+. V , − h 20 ms 8 holding potential. (D) current–voltage relationships +TTX 2+ −10 of TTX-sensitive current in the presence of Co at 0mV a holding potential of −70 mV (᭺) and at a C depolarised holding potential of −40 mV (᭹). Vh −70 mV Current in D is normalized for cell capacitance. +Co2+ squid, cuttlefish and Eledone were recorded at a holding on depolarization (Fig. 6). This current was blocked by the potential of −70 mV. Mean whole-cell capacitances for squid addition of 5 mmol l−1 Co2+ (see Fig. 5). In squid, the peak were 152.78±18.6 pF−1 (N=16), for cuttlefish 68.01±7.94 pF−1 current was −10.95±0.79 pA. The midpoint potential was (N=12) and for Eledone 163.5±44.43 pF−1 (N=7) (mean ± +10±3.27 mV and it had a mean activation point of S.E.M.). Whole-cell capacitances measured in squid and −35.71±2.02 mV (N=7). In cuttlefish, the peak current was Eledone were significantly different from those measured in −5.32±0.81 pA and had a midpoint potential of +2±2.9 mV. cuttlefish (t=3.73, t=−2.74, respectively, P<0.005). This current activated at a mean value of −23.75±4.6 mV Capacitances measured in squid and Eledone were not, (N=10). In Eledone, the peak current was −0.69±0.03 pA, with however, significantly different. Depolarising steps elicited a midpoint potential of +21.25±2.95 mV and it activated at currents (as seen in Figs 5 and 6) when the external solution −12.5±7.5 mV (N=5) (Fig. 6C; all values are mean ± S.E.M.). −1 −1 contained 2 mmol l CaCl2. The currents seen in squid muscle Application of 10 µmol l nifedipine suppressed the inward preparations consisted of two components: an initial fast- current in squid, Eledone and cuttlefish myocytes (Fig. 6B). activating inward current and a second more slowly activating These properties indicate that this more slowly activating inward current (Fig. 5), while in cuttlefish and Eledone muscle inward current is an L-type, dihydropyridine (DHP)-sensitive only the slowly activating inward current was observed Ca2+ current. (Fig. 6). Outward currents were not seen, presumably because any voltage-activated K+ currents were blocked by the Cs+ in the internal solution. Discussion This study describes the membrane properties and Fast-activating inward currents contractile responses of muscle fibres from the mantle of Fast-activating inward currents were only seen in squid . Specifically, there are quantitative physiological myocytes and were observed in all muscle fibres studied (N=20 differences between central zone fibres isolated from squid, from 11 animals). Addition of 2 µmol l−1 TTX to the bath cuttlefish and Eledone mantle. completely blocked this inward current (Fig. 5B), as did holding the cell potential at relatively depolarised values Ca2+ currents (−40 mV, Fig. 5D). As this current was not blocked by the The slowly activating inward current recorded from squid, addition of Co2+ (Fig. 5D), it could be studied without cuttlefish and Eledone mantle was insensitive to TTX, blocked interference from other more slowly activating inward currents by Co2+ and suppressed by nifedipine. This sustained current (see below and Fig. 5). Under these conditions, the is identified as an L-type, DHP-sensitive Ca2+ current. It seems current–voltage relationships reversed at approximately +30 to likely that Ca2+ influx through this channel drives +40 mV, as expected for a Na+ current (Fig. 5). Na+ currents excitation–contraction coupling in cephalopod mantle muscle were not observed in material from cuttlefish (N=12) or since the contractile responses of both squid and cuttlefish Eledone (N=7). slices were reversibly abolished in the absence of extracellular Ca2+. This is unsurprising because it is known that the presence Ca2+ currents of external Ca2+ and Ca2+ entry through voltage-activated L- In squid (after blocking the Na+ current with TTX), Eledone type Ca2+ channels is necessary for functional EC coupling of and cuttlefish, a slowly activating inward current was observed invertebrate striated muscle fibres (see Inoue et al. 1994). EC coupling in cephalopod mantle muscle 3039

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− −50 to +50 mV 0.4 2000 pA +10 mV ) 1 20 ms − − Vh 70 mV Vh −70 mV −0.8 (pA pF I Fig. 6. (A) Typical whole-cell voltage-clamp currents from squid, cuttlefish and Eledone cirrhosa mantle muscle. Currents are normalized for cell capacitance. (B) Peak whole-cell currents shown before (solid line) and after (broken line) the application of 10 µmol l−1 nifedipine (not the same preparation as in A). Vh, holding potential. (C) Current–voltage (I–V) relationships of whole-cell currents normalized for capacitance in squid (N=7), cuttlefish (N=10) and Eledone (N=5) muscle. Values are means ± S.E.M. Squid currents were measured in the presence of 2 µmol l−1 tetrodotoxin.

Na+ currents in squid muscle observable effect on slice length, indicating that the cut radial A fast inward current was recorded only from squid mantle fibres were damaged and unable to shorten in response to the muscle fibres. This current is likely to be carried by Na+, as it presence of the neurotransmitter (Milligan et al. 1997). In the was blocked by TTX, inactivated by depolarising holding earlier study of Gilly et al. (1996), both circular and radial fibres potentials and reversed at approximately +30 to +40 mV. Na+ were sampled, as the muscle was dissociated from a vertical currents were identified in every squid mantle muscle fibre ‘slice’. These observations imply that the outer and inner fibres examined. In contrast, in a recent study, a subpopulation do not possess Na+ channels. One reservation with this view, representing only 50 % of the total fibres was found to possess however, is that when we examined vertical slices from several Na+ channels (Gilly et al. 1996). This difference can be adult squid (data not shown) Na+ currents were detected in all attributed to a number of possible factors. The fibres in our fibres. At this stage, we cannot rule out another explanation, study were dissociated from 0.1 mm muscle slices derived from which is that there may be ontogenetic differences in muscle the central zone of mitochondria-poor fibres. The mitochondria- fibre channel expression. The study of Gilly et al. (1996) was rich superficial fibres of the inner and outer layers were not carried out on juvenile squid Loligo opalescens whereas our sampled and, because of the plane of section, it is probable that study was carried out on adult A. subulata. Species differences the thin muscle slices prepared for dissociation contained no cannot be discounted either for, as we report in the present intact radial fibres. Radial fibres have been shown to be study, there are striking differences in channel expression in the sensitive to acetylcholine (Bone and Howarth, 1980) and, in a mantle muscle of different cephalopods. Further experiments previous study using slices, acetylcholine superfusion had no are required to resolve these issues. 3040 C. M. ROGERS AND OTHERS

Functional implications of Na+ channels in squid of excitation–contraction (EC) coupling in invertebrates. It has The biomechanical experiments support the idea that the been shown that invertebrate skeletal muscle EC coupling is Na+ current in squid fibres is associated with enhanced quantitatively different from that of vertebrate striated muscle propagation of excitation through the mantle. First, in g-ASW, (Inoue et al. 1994) in that it is presumed to be driven by Ca2+ squid preparations were maximally activated at a lower current influx alone rather than by voltage-sensitive Ca2+ release. In strength than cuttlefish preparations of similar thickness. vertebrate skeletal muscle, Na+ channels depolarise the Second, in Na+-free ASW, higher current strengths were membrane, activating the L-type Ca2+ channel voltage sensor required to elicit a supramaximal response in squid and, subsequently, the intracellular Ca2+ release channel preparations. Third, in contrast to cuttlefish preparations, the (ryanodine receptor) on the sarcoplasmic reticulum, releasing current–response relationships of squid preparations were Ca2+ from internal stores. The activation of the L-type Ca2+ insensitive to slice thickness. This means that propagation is current is extremely slow in vertebrate skeletal muscle (of the all-or-nothing rather than electrotonic in squid mantle muscle, order of seconds), so very little Ca2+ enters during the action which appears to activate more readily (at least in response to potential. This may be contrasted with the situation in squid direct stimulation) than cuttlefish mantle muscle. Brown muscle, where the activation of the L-type Ca2+ channel is fast (1991), cited in Bone et al. (1995), recorded rapid overshooting (milliseconds) and there may be sufficient Ca2+ entry during spike potentials in central mantle fibres of squid during the to activate contraction and/or Ca2+-induced stimulation of the stellar nerves. It is possible that these spikes Ca2+ release. Because of the fast activation of the Ca2+ current, represent Na+-based, propagating action potentials, absent in it seems unlikely that squid operate a ‘vertebrate skeletal cuttlefish fibres, that transmit excitation effectively from fibre muscle type’ of voltage-induced Ca2+ release, although this to fibre. Previous experiments have shown that central fibres idea has yet to be tested directly. in both squid and cuttlefish are dye-coupled (Bone et al. 1995) The presence of Na+ channels has been reported previously and electrically coupled (Milligan et al. 1997) and that this in the muscles of invertebrates, including the protochordate coupling is important for effective activation of tissue slices Amphioxus (Branchiostoma lanceolatum) (Hagiwara and (Milligan et al. 1997). It seems unlikely, therefore, that every Kikodoro, 1971), the chaetognathe Sagitta elegans (Schwartz fibre needs to be directly innervated in squid and cuttlefish, but and Stuhmer, 1984) and the coelenterate Chelophyes that excitation may spread from innervated cells to non- appendiculata (Chain et al. 1981). However, the striated innervated cells via gap junctions. Propagated action potentials muscles of other invertebrates examined have been found to in squid would certainly effect this spread more efficiently than possess only voltage-activated Ca2+ channels, as in the example electrotonic potentials. here of the cuttlefish and octopod (see also Bézina et al. 1994). We can conclude that there are at least three possible Absence of Na+ currents in other cephalopods arrangements of channels for EC coupling. In vertebrate We were unable to detect Na+ currents in fibres isolated skeletal muscle, there is a Na+ and Ca2+ channel voltage- from cuttlefish or Eledone mantle, and our mechanical sensitive release mechanism where depolarization of the muscle experiments confirm that the contractile behaviour of membrane activates a propagating Na+-based action potential cuttlefish fibres is independent of the presence of external that subsequently activates L-type Ca2+ channels which, in turn, Na+. In cuttlefish, the organization of the mantle musculature gate direct release of Ca2+ from the sarcoplasmic reticulum. In is similar to that described for squid, and the cells studied are vertebrate cardiac and some invertebrate striated muscle, there also likely to be derived from the central zone of is a Na+ and Ca2+ channel arrangement where the Na+-based mitochondria-poor fibres. In Eledone, the structure of the action potential opens L-type Ca2+ channels and allows Ca2+ mantle is more complex, with several intricate layers of fibres influx followed by Ca2+-induced Ca2+ release. In other (Wells, 1978), and so it was not possible to isolate a specific invertebrate striated muscles, only L-type Ca2+ channels are population of cells. Nevertheless, we did not observe Na+ present and, in these, contraction may be graded by currents in any of the dissociated fibres that we examined. It depolarisation of the muscle and opening of Ca2+ channels. is remarkable that the distribution of ion channels and the Our slice experiments show that the central zone fibres in morphology of the mantle and its innervation in these animals squid have Na+ channels, and the existence of Na+ channels apparently reflects their lifestyles. Squid are fast-swimming allows a rapid all-or-nothing response in these muscle fibres. pelagic predators, capable of rapid accelerations (O’Dor, In cuttlefish and Eledone cirrhosa, Na+ channels are absent 1988). Cuttlefish and Eledone are benthic, relatively slow- from the central zone. This striking difference in muscle moving predators and their mantle muscle reflects this fact. physiology in closely related species highlights the fact that There are other specialised muscle fibres in cephalopods there can be adaptive radiation of physiological processes which would be interesting to examine from this point of within phyla and that remarkable biochemical and view, for instance the rapidly activating cross-striated physiological specialisations have been acquired during muscles of the tentacle (Kier, 1991). cephalopod evolution.

Implications for EC coupling We would like to thank the director and staff of the Marine The above results have implications for our understanding Biological Association for the provision of animals and EC coupling in cephalopod mantle muscle 3041 facilities. We would also like to thank Quentin Bone for his fibres of squid mantle. Biol. Bull. mar. biol. Lab., Woods Hole 191, helpful comments on the manuscript. L.N. was supported by 337–340. an MBA bursary, C.M.R., B.J.M. and E.R.B. are supported by HAGIWARA, S. AND KIKODORO, Y. (1971). Na and Ca components of the BBSRC. action potential in Amphioxus muscle cells. J. Physiol., Lond. 219, 217–232. INOUE, I., TSUTSUI, I., BONE, Q. AND BROWN, E. R. (1994). Evolution References of skeletal muscle excitation–contraction coupling and the BÉZINA, V., EVANS, C. G. AND WEISS, K. R. (1994). Characterisation appearance of dihydropyridine-sensitive intramembrane charge of the membrane ion currents of a model molluscan muscle, the movement. Proc. R. Soc. Lond. B 225, 181–187. accessory closer muscle of Aplysia californica. III. KIER, W. M. (1991). Squid cross-striated muscle: The evolution of a Depolarisation-activated Ca current. J. Neurophysiol. 71, specialised muscle fibre type. Bull. mar. Sci. 49, 389–403. 2126–2138. MENDEZ, J. AND KEYS, K. (1960). Density and composition of BONE, Q., BROWN, E. R., NICHOLSON, D. AND TRAVERS, G. (1994). On mammalian muscle. Metabolism 9, 184–188. the respiratory flow in the cuttlefish, Sepia officinalis. J. exp. Biol. MILLIGAN, B. J., CURTIN, N. A. AND BONE, Q. (1997). Contractile 194, 153–165. properties of obliquely striated muscle from the mantle of squid BONE, Q., BROWN, E. R. AND USHER, M. (1995). The structure and (Alloteuthis subulata) and cuttlefish (Sepia officinalis). J. exp. Biol. physiology of cephalopod muscle fibres. In Cephalopod 200, 2425–2436. Neurobiology (ed. N. J. Abbott, R. Williamson and L. Maddock), MOMMSEN, T. P., BALLANTYNE, J., MACDONALD, D., GOSLINE, J. AND pp. 301–309. Oxford: Oxford University Press. HOCHACHKA, P. W. (1981). Analogues of red and white muscle in BONE, Q. AND HOWARTH, J. V. (1980). The role of L-glutamate in squid mantle. Proc. natn. Acad. Sci. U.S.A. 78, 3274–3278. neuromuscular transmission in some molluscs. J. mar. biol. Ass. O’DOR, R. K. (1988). The forces acting on swimming squid. J. exp. U.K. 60, 619–626. Biol. 137, 421–442. BONE, Q., PULSFORD, A. AND CHUBB, A. D. (1981). Squid mantle OTIS, T. S. AND GILLY, W. F. (1990). Jet-propelled escape in the squid muscle. J. mar. biol. Ass. U.K. 61, 321–342. Loligo opalescens: concerted control by giant and non-giant motor BROWN, E. R. AND BONE, Q. (1991). Repetitive stimulation of the axon pathways. Proc. natn. Acad. Sci. U.S.A. 87, 2911–2915. squid giant axon exerts graded control over mantle tension. J. mar. PROSSER, C. L. AND YOUNG, J. Z. (1937). Responses of muscles of the biol. Ass. U.K. 71, 732. squid to repetitive stimulation of the giant nerve fibre. Biol. Bull. BROWN, E. R., USHER, M. L. AND BONE, Q. (1991). Physiological mar. biol. Lab., Woods Hole 73, 237–241. properties of squid mantle muscle bundles. J. mar. biol. Ass. U.K. SCHWARTZ, L. AND STUHMER, W. (1984). Voltage-dependent sodium 71, 732–733. channels in an invertebrate striated muscle. Science 225, 532–535. CHAIN, B. M., BONE, Q. AND ANDERSON, P. A. V. (1981). WELLS, M. J. (1978). Octopus – Physiology and Behaviour of an Electrophysiology of a myoid epithelium in Chelophyes Advanced Invertebrate. London: Chapman & Hall. (Coelenterata: Siphonophora). J. comp. Physiol. A 143, WILSON, D. M. (1960). The control of movement in cephalopods. J. 329–338. exp. Biol. 37, 57–72. GILLY, W. F., PREUSS, T. AND MCFARLANE, A. B. (1996). All-or-none YOUNG, J. Z. (1938). The functioning of the giant nerve fibres of the contraction and sodium channels in a subset of circular muscle squid. J. exp. Biol. 15, 170–185.